Radioiodinated Fatty Acids

ABSTRACT

The present invention provides novel radioiodinated fatty acids. Also provided are methods of preparation of said radioiodinated fatty acids from non-radioactive precursors, as well as radiopharmaceutical compositions comprising such radioiodinated fatty acids. The invention also provides in vivo imaging methods using the radioiodinated fatty acids.

FIELD OF THE INVENTION

The present invention provides novel radioiodinated fatty acids. Alsoprovided are methods of preparation of said radioiodinated fatty acidsfrom non-radioactive precursors, as well as radiopharmaceuticalcompositions comprising such radioiodinated fatty acids. The inventionalso provides in vivo imaging methods using the radioiodinated fattyacids.

BACKGROUND TO THE INVENTION

Under normal conditions, the human heart derives more than 60% of itsenergy from the oxidative metabolism of long chain fatty acids. In theischaemic myocardium, however, oxidative metabolism of free fatty acidsis suppressed, and anaerobic glucose metabolism predominates. Metabolicimaging can therefore provide useful information in the diagnosis andmonitoring of various forms of heart disease.

Fatty acids have been radiolabelled with ¹¹C and ¹⁸F for PET imaging,and ¹²³I and ^(99m)Tc for SPECT radiopharmaceutical imaging [Eckelman etal, J.Nucl. Cardiol., 14, S100-S109 (2007)]. Eckelman et at stress thatradiolabelling with an isotope other than ¹¹C is in fact labelling afatty acid analogue, and that care is needed that the substituent doesnot affect the ability of the analogue to trace important steps of themetabolic pathway.

Taki et at [Eur. J. Nucl. Med. Mol. Imaging, 34, S34-S48 (2007)] pointout that early radioiodinated fatty acid analogues based on iodo-alkylsubstituents were found to suffer significant in vivo metabolicdeioidination. Radioiodinated fatty acid analogues incorporatingiodo-phenyl moieties such as ¹²³I-BMIPP and ¹²³I-IPPA have, however,become established agents for such metabolic imaging (Taki et al, citedabove):

-   -   where I*=¹²³I.

The applications of “click chemistry” in biomedical research, includingradiochemistry, have been reviewed by Nwe et at [Cancer Biother.Radiopharm., 24(3), 289-302 (2009)]. As noted therein, the main interesthas been in the PET radioisotope ¹⁸F (and to a lesser extent ¹¹C), plus“click to chelate” approaches for radiometals suitable for SPECT imagingsuch as ^(99m)Tc or ¹¹¹In. ¹⁸F click-labelling of targeting peptides,giving products incorporating an ¹⁸F-fluoroalkyl-substituted triazolehave been reported by Li et at [Bioconj. Chem., 18(6), 1987-1994 (2007)]and Hausner et at [J. Med. Chem., 51(19), 5901-5904 (2008)].

WO 2006/067376 discloses a method for labelling a vector comprisingreaction of a compound of formula (I) with a compound of formula (II):

or,a compound of formula (III) with a compound of formula (IV)

in the presence of a Cu(I) catalyst, to give a conjugate of formula (V)or (VI) respectively:

wherein L1, L2, L3, and L4 are each Linker groups;

-   -   R* is a reporter moiety which comprises a radionuclide.

R* of WO 2006/067376 is a reporter moiety which comprises a radionuclidefor example a positron-emitting radionuclide. Suitable positron-emittingradionuclides for this purpose are said to include ¹¹C, ¹⁸F, ⁷⁵Br, ⁷⁶Br,¹²⁴I, ⁸²Rb, ⁶⁸Ga, ⁶⁴Cu and ⁶²Cu, of which ¹¹C and ¹⁸F are preferred.Other useful radionuclides are stated to include ¹²³I, ¹²⁵I, ¹³¹I,²¹¹At, ^(99m)Tc, and ¹¹¹In.

WO 2007/148089 discloses a method for radiolabelling a vector comprisingreaction of a compound of formula (I) with a compound of formula (II):

or, a compound of formula (III) with a compound of formula (IV):

in the presence of a Cu(I) catalyst to give a conjugate of formula (V)or (VI) respectively:

wherein:

-   -   L1, L2, L3, and L4 are each Linker groups;    -   R* is a reporter moiety which comprises a radionuclide.

In both WO 2006/067376 and WO 2007/148089, metallic radionuclides arestated to be suitably incorporated into a chelating agent, for exampleby direct incorporation by methods known to the person skilled in theart.

WO 2006/116629 (Siemens Medical Solutions USA, Inc.) discloses a methodof preparation of a radiolabelled ligand or substrate having affinityfor a target biomacromolecule, the method comprising:

-   -   (a) reacting a first compound comprising        -   (i) a first molecular structure;        -   (ii) a leaving group;        -   (iii) a first functional group capable of participating in a            click chemistry reaction; and optionally,        -   (iv) a linker between the first functional group and the            molecular structure, with a radioactive reagent under            conditions sufficient to displace the leaving group with a            radioactive component of the radioactive reagent to form a            first radioactive compound;    -   (b) providing a second compound comprising        -   (i) a second molecular structure;        -   (ii) a second complementary functional group capable of            participating in a click chemistry reaction with the first            functional group, wherein the second compound optionally            comprises a linker between the second compound and the            second functional group;    -   (c) reacting the first functional group of the first radioactive        compound with the complementary functional group of the second        compound via a click chemistry reaction to form the radioactive        ligand or substrate; and    -   (d) isolating the radioactive ligand or substrate.

WO 2006/116629 teaches that the method therein is suitable for use withthe radioisotopes: ¹²⁴I, ¹⁸F, ¹³N and ¹⁵O with preferred radioisotopesbeing: ¹⁸F, ¹¹C, ¹²³I, ¹²⁴I, ¹²⁷I, ¹³¹I, ⁷⁶Br, ⁶⁴ Cu, ^(99m)Tc, ⁹⁰Y,⁶⁷Ga, ⁵¹Cr, ¹⁹²Ir, ⁹⁹Mo, ¹⁵³SM and ²⁰¹Tl. WO 2006/116629 teaches thatother radioisotopes that may be employed include: ⁷²As, ⁷⁴As, ⁷⁵Br,⁵⁵Co, ⁶¹Cu, ⁶⁷Cu, ⁶⁸Ga, ⁶⁸Ge, ¹²⁵I, ¹³²I, ¹¹¹In, ⁵²Mn, ²⁰³Pb and ⁹⁷Ru.WO 2006/116629 does not, however, provide any specific teaching on howto apply the method to the radioiodination of biological molecules.

WO 2010/129572 describes PET radiotracers for imaging fatty acidmetabolism and storage having one of the following formulae:

-   -   where: n is 10-24, m is 1-10 and X is a halogen.

WO 2010/129572 teaches that at least one atom of the above chemicalstructures can be a radionuclide, preferably a positron-emittingradioisotope. ¹⁸F is the main radioisotope described. The structuresshown would not be expected to be suitable for labelling withradioiodine, since if X were to be iodine that requires an iodoalkylgroup, and such groups are known to be unstable with respect todeiodination in vivo.

Kim et at [Bioconj. Chem., 20(6), 1139-1145 (2009) disclose ¹⁸F-labelledfatty acid analogues for PET imaging of myocardial metabolism:

The ¹⁸F-fatty acids were prepared via click cycloaddition, wherein an¹⁸F-alkyne was coupled to an azido-fatty acid, to generate the triazolering.

PET imaging with ¹⁸F typically requires the availability of a cyclotronfacility on the same site as the hospital, since ¹⁸F has a shorthalf-life (110 minutes) and the desired radiotracer needs to besynthesised. The availability of cameras suitable for PET imaging isconsequently much less widespread than SPECT cameras. There is thereforestill a need for alternative radioiodinated fatty acids suitable formore routine clinical imaging, especially using SPECTradiopharmaceutical imaging.

The longer half-life of ¹²³I compared to ¹⁸F enables the cyclotron forits production to be up to one day's transport time from the end user.This makes it possible for a single cyclotron to be able to supply acontinent rather than a city, as is the case with ¹⁸F fluorineproduction.

THE PRESENT INVENTION

The present invention provides radioiodinated fatty acid analoguescomprising triazole or isoxazole rings. The triazole and isoxazole ringsdo not hydrolyse and are highly stable to oxidation and reduction,meaning that the labelled fatty acid has high in vivo stability. Thetriazole ring is also comparable to an amide in size and polarity. Thetriazole and isoxazole rings of the fatty acids of Formula (I) of thepresent invention are not expected to be recognized by thyroiddeiodination enzymes known to metabolise iodo-tyrosine more rapidly thaniodobenzene, and are thus expected to be sufficiently stable in vivo forradiopharmaceutical imaging and/or radiotherapy.

When the iodine isotope is ¹²³I or ¹³¹I, the fatty acids of the presentinvention have the advantage that they are suitable for SPECT imaging,and hence have a wider clinical potential than PET agents, due to thewider availability of gamma cameras. The radioiodinated fatty acidanalogues can be synthesised readily using either click chemistry, ororganometallic precursors. Mild reaction conditions are required for thesynthesis of the carbon iodine bond and this enables sensitive moleculesto be radioiodinated. In general radiofluorination requires much moreforcing conditions rendering it unsuitable for very sensitive molecules.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a radioiodinated fattyacid of Formula (I):

-   -   where:    -   R¹ and R² are independently H or C₁₋₂ alkyl;    -   Y is a Y¹ or Y² group:

-   -   p and q are each independently integers of value 0 to 10 which        are chosen such that [p+q] is in the range 10 to 16;    -   L¹ is a linker group of formula -(A)_(n)- where n is an integer        of value 0 to 3, and each A group is independently chosen from        —CH₂—, —O—, —S— and —C₆H₄— with the proviso that L¹ does not        comprise —O—O—, —S—S— or —O—S— linkages;    -   I* is a radioisotope of iodine.

The term “radioiodinated” has its conventional meaning, i.e. aradiolabelled compound wherein the radioisotope used for theradiolabelling is a radioisotope of iodine. The term “radioisotope ofiodine” has its conventional meaning, i.e. an isotope of the elementiodine that is radioactive. Suitable such radioisotopes include: ¹²³I,¹²⁴I, ¹²⁵I and ¹³¹I.

The term “fatty acid” has its conventional meaning, i.e. a monobasicaliphatic carboxylic acid, typically having at least a 10-carbon chain.

Preferred Aspects.

Preferred radioisotopes of iodine for use in the present invention arethose suitable for medical imaging in vivo using PET or SPECT,preferably ¹²³I, ¹²⁴I or ¹³¹I, more preferably ¹²³I or ¹²⁴I, mostpreferably ¹²³I.

A preferred radioiodinated fatty acid of the first aspect is where Y isY¹, i.e. the radioiodine isotope is attached to a triazole ring.

In Formula (I), preferably at least one of R¹ and R² is C₁₋₂ alkyl, morepreferably methyl. Most preferably, le is CH₃ and R² is H.

In Formula (I), L¹ is preferably chosen from —CH₂—, —O— and —S—. InFormula (I), n is preferably 0. In Formula (I), [p+q] is preferably inthe range 10 to 14. When n=0, [p+q] is preferably in the range 11 to 14,more preferably 11 to 13.

The radioiodinated fatty acids of Formula (I) may be obtained asdescribed in the second or third aspects (below). The preparation methodof the second aspect (via Precursor IA) is preferred, since thatcomprises only a single step in which radioactive manipulations areinvolved (a single step iododemetallation reaction)—thus minimising theradiation dose to the operator.

Included within the scope of the first aspect is an imaging agent whichcomprises the radioiodinated fatty acid of Formula (I). By the term“imaging agent” is meant a compound suitable for imaging the mammalianbody. Preferably, the mammal is an intact mammalian body in vivo, and ismore preferably a human subject. Preferably, the imaging agent can beadministered to the mammalian body in a minimally invasive manner, i.e.without a substantial health risk to the mammalian subject when carriedout under professional medical expertise. Such minimally invasiveadministration is preferably intravenous administration into aperipheral vein of said subject, without the need for local or generalanaesthetic. The imaging agents of the first aspect are preferably usedas radiopharmaceutical compositions, as described in the fourth aspect(below).

In a second aspect, the present invention a method of preparation of theradioiodinated fatty acid of Formula (I) of the first aspect, where saidmethod comprises:

-   -   (i) provision of a precursor of Formula (IA)

-   -   -   where:        -   R¹, R², L¹, p and q are as defined in any one of claims 1 to            6;        -   Visa Y^(1a) or Y^(2a) group:

-   -   -   -   wherein Q is R^(a) ₃Sn— or KF₃B—, where each R^(a) is                independently C₁₋₄ alkyl;

    -   (ii) reaction of said precursor with radioactive iodide ion in        the presence of an oxidising agent to give the radioiodinated        fatty acid of Formula (I).

Preferred embodiments of R¹, R², L¹, p and q and I* in the second aspectare as defined in the first aspect.

In Y^(a), when Q is KF₃B—, that corresponds to a potassiumtrifluoroborate derivative as described below.

By the term “oxidising agent” is meant an oxidant capable of oxidisingiodide ion to form the electrophilic species (HOT, H₂OI), wherein theactive iodinating agent is I⁺. Suitable oxidising agents are describedby Bolton [J. Lab. Comp. Radiopharm., 45, 485-528 (2002)], and Eerselset al [J. Lab. Comp. Radiopharm., 48, 241-257 (2005)] and includeperacetic acid and N-chloro compounds, such as chloramine-T, iodogen,iodogen tubes and succinimides. Preferred oxidising agents are peraceticacid (which is commercially available) at pH ca. 4, and hydrogenperoxide/aqueous HCl at pH ca. 1. Iodogen tubes are commerciallyavailable from Thermo Scientific Pierce Protein Research Products.

By the term “radioactive iodide ion” is meant a radioisotope of iodine(as defined above), in the chemical form of iodide ion (I⁻).

When Q is R^(a) ₃Sn—, the radioiodination method of the third aspect iscarried out as described by Bolton [J. Lab. Comp. Radiopharm., 45,485-528 (2002)] and Eersels et at [J. Lab. Comp. Radiopharm., 48,241-257 (2005)]. The organotin precursors are prepared as described byAli et at [Synthesis, 423-445 (1996)].

When Q is KF₃B—, the radioiodination reaction method of the third aspectcan be carried out as described by Kabalka et al [J. Lab. Comp.Radiopharm., 48, 359-362 (2005)], who use peracetic acid as theoxidising agent. Precursors where Q is KF₃B— can be obtained from thecorresponding alkyne as described by Kabalka et at [J. Lab. Comp.Radiopharm., 48, 359-362 (2005) and, J. Lab. Comp. Radiopharm., 49,11-15 (2006)]. The potassium trifluoroborate precursors are stated to becrystalline solids, which are stable to both air and water.

The radioiodination reaction of the second aspect may be effected in asuitable solvent, for example acetonitrile, a C₁₋₄ alkylalcohol,dimethylformamide, tetrahydrofuran (THF), or dimethylsulfoxide, ormixtures thereof, or aqueous mixtures thereof, or in water. Aqueousbuffers can also be used. The pH will depend on the oxidant used, andwill typically be pH 0 to 1 when eg. hydrogen peroxide/aqueous acid isused, or in the range pH 6-8 when iodogen or iodogen tubes are used. Theradioiodination reaction temperature is preferably 10 to 60° C., morepreferably at 15 to 50° C., most preferably at ambient temperature(typically 15-37° C.). Organic solvents such as acetonitrile or THFand/or the use of more elevated temperature may conveniently be used tosolubilise any precursors of Formula (TB) which are poorly soluble inwater.

The precursor of Formula (IA) is suitably non-radioactive, so can beprepared and purified by conventional means without the need forradiation handling safety precautions.

In a third aspect, the present invention provides a method ofpreparation of the radioiodinated fatty acid of Formula (I) as definedin the first aspect, where said method comprises:

-   -   (i) provision of a precursor of Formula (IB)

-   -   -   where:        -   R¹, R², L¹, p and q are as defined in any one of claims 1 to            6;        -   Y^(b) is a Y^(th) or Y^(2b) group:

-   -   (ii) reaction of said precursor with a compound of Formula (II):

-   -   -   in the presence of a click cycloaddition catalyst, to give            the radioiodinated fatty acid of Formula (I) via click            cycloaddition,            -   wherein I* is a radioisotope of iodine, as defined in                claim 1 or claim 2.

In Formula (IB), Y can be either an azide substituent (Y=Y^(1a)), or anisonitrile oxide substituent (Y=Y^(2a)).

Preferred embodiments of R¹, R², L¹, p and q and I* in the third aspectare as defined in the first aspect.

By the term “click cycloaddition catalyst” is meant a catalyst known tocatalyse the click (alkyne plus azide) or click (alkyne plus isonitrileoxide) cycloaddition reaction. Suitable such catalysts are known in theart for use in click cycloaddition reactions. Preferred such catalystsinclude Cu(I), and are described below. Further details of suitablecatalysts are described by Wu and Fokin [Aldrichim. Acta, 40(1), 7-17(2007)] and Meldal and Tornoe [Chem. Rev., 108, 2952-3015 (2008)]. Theapplications of “click chemistry” in biomedical research, includingradiochemistry, have been reviewed by Nwe et al [Cancer Biother.Radiopharm., 24(3), 289-302 (2009)].

A preferred click cycloaddition catalyst comprises Cu(I). The Cu(I)catalyst is present in an amount sufficient for the reaction toprogress, typically either in a catalytic amount or in excess, such as0.02 to 1.5 molar equivalents relative to the compound of Formula (Ia)or (Ib). Suitable Cu(I) catalysts include Cu(I) salts such as Cul or[Cu(NCCH₃)₄][PF₆], but advantageously Cu(II) salts such as copper (II)sulphate may be used in the presence of a reducing agent to generateCu(I) in situ. Suitable reducing agents include: ascorbic acid or a saltthereof for example sodium ascorbate, hydroquinone, metallic copper,glutathione, cysteine, Fe²⁺, or Co²⁺. Cu(I) is also intrinsicallypresent on the surface of elemental copper particles, thus elementalcopper, for example in the form of powder or granules may also be usedas catalyst. Elemental copper, with a controlled particle size is apreferred source of the Cu(I) catalyst. A more preferred such catalystis elemental copper as copper powder, having a particle size in therange 0.001 to 1 mm, preferably 0.1 mm to 0.7 mm, more preferably around0.4 mm. Alternatively, coiled copper wire can be used with a diameter inthe range of 0.01 to 1.0 mm, preferably 0.05 to 0.5 mm, and morepreferably with a diameter of 0.1 mm. The Cu(I) catalyst may optionallybe used in the presence of bathophenanthroline, which is used tostabilise Cu(I) in click chemistry.

In the method of the third aspect, the compound of Formula (II) mayoptionally be generated in situ by deprotection of a compound of Formula(IIa):

-   -   wherein M¹ is an alkyne-protecting group, and I* is as defined        for Formula (II). Preferred aspects of I* in Formula (IIa), are        as described for Formula (II).

By the term “protecting group” is meant a group which inhibits orsuppresses undesirable chemical reactions, but which is designed to besufficiently reactive that it may be cleaved from the functional groupin question under mild enough conditions that do not modify the rest ofthe molecule. After deprotection the desired product is obtained.Suitable alkyne protecting groups are described in Protective Groups inOrganic Synthesis, Theodora W. Greene and Peter G. M. Wuts, Chapter 8,pages 927-933, 4^(th) edition (John Wiley & Sons, 2007), and include: antrialkylsilyl group where each alkyl group is independently C₁₋₄ alkyl;an aryldialkylsilyl group where the aryl group is preferably benzyl orbiphenyl and the alkyl groups are each independently C₁₋₄ alkyl;hydroxymethyl or 2-(2-hydroxypropyl). A preferred such alkyne protectinggroup is trimethylsilyl. The protected iodoalkynes of Formula (IIa) havethe advantages that the volatility of the radioactive iodoalkyne can becontrolled, and that the desired alkyne of Formula (II) can be generatedin a controlled manner in situ so that the efficiency of the reactionwith the precursor of Formula (IA) is maximised.

The click cycloaddition method of the second aspect may be effected in asuitable solvent, for example acetonitrile, a C₁₋₄ alkylalcohol,dimethylformamide, tetrahydrofuran, or dimethylsulfoxide, or aqueousmixtures of any thereof, or in water. Aqueous buffers can be used in thepH range of 4-8, more preferably 5-7. The reaction temperature ispreferably 5 to 100° C., more preferably at 75 to 85° C., mostpreferably at ambient temperature (typically 15-37° C.). The clickcycloaddition may optionally be carried out in the presence of anorganic base, as is described by Meldal and Tornoe [Chem. Rev. 108,2952, Table 1 (2008)].

A preferred precursor of Formula (IB) has Y^(b)=Y^(ib). One reason isthat the isonitrile oxides are typically less stable than azides.Consequently, whilst the azide of Formula (IB, Y^(b)=Y^(1b)) can beisolated and purified, the isonitrile oxide of Formula (IB,Y^(b)=Y^(2b)) will typically need to be generated in situ.

The non-radioactive precursor compound of Formula (IB), where Y^(b) isY^(1b) (azido derivatives) may be prepared by either:

-   -   (i) reaction of the corresponding bromo-fatty acid with sodium        azide;    -   (ii) conversion of the corresponding hydroxy-fatty acid to a        tosylate or mesylate derivative, and subsequent reaction with        sodium azide.

Further details are provided by Kim et al [Bioconj. Chem., 20(6),1139-1145 (2009)], and Kostiuk et al [Meth. Enzymol., 457, 149-165(2009)]. Many functionalised fatty acids are commercially available.

The non-radioactive precursor compound of Formula (IB), where Y^(b) isY^(2b) (isonitrile oxide derivatives) may be prepared by the methodsdescribed by Ku et al [Org. Lett., 3(26), 4185-4187 (2001)], andreferences therein. Thus, they are typically generated in situ bytreatment of an alpha-halo aldoxime with an organic base such astriethylamine. A preferred method of generation, as well as conditionsfor the subsequent click cyclisation to the desired isoxazole aredescribed by Hansen et al [J. Org. Chem., 70(19), 7761-7764 (2005)].Hansen et al generate the desired alpha-halo aldoxime in situ byreaction of the corresponding aldoxime with chloramine-T trihydrate, andthen dechlorinating this with sodium hydroxide. The correspondingaldoxime is prepared by reacting the corresponding aldehyde withhydroxylamine hydrochloride at pH 9-10. See also K. B. G. TorsellNitrile Oxides, Nitrones and Nitronates in Organic Synthesis [VCH, NewYork (1988)]. Ω-aldehyde functionalised fatty acids are readilyaccessible by the oxidation of the corresponding alcohol in a Swernoxidation. The alcohols are generally commercially available but arealso accessible by ozone oxidation to the ozonide of the correspondingunsatuturated fatty acid followed by borohydride reduction to thealcohol. A very wide range of unsaturated fatty acids are available fromnatural product sources.

Included within the scope of this third aspect, is the option of usingan aldoxime precursor, wherein instead of Y^(2b), Y^(b) is chosen to be(HO)N═CH—, so that the isonitrile oxide (Y^(b)=Y^(2b)) is generated insitu. The steps involved can be carried out sequentially without workup.The resulting nitrile oxide is not particularly stable and is best usedimmediately, preferably in situ without workup.

The preparation methods of the second and third aspects are preferablycarried out in an aseptic manner, such that the product of Formula (I)is obtained as a radiopharmaceutical composition. Thus, the method iscarried out under aseptic manufacture conditions to give the desiredsterile, non-pyrogenic radiopharmaceutical product. It is preferredtherefore that the key components, especially any parts of the apparatuswhich come into contact with the product of Formula (I) (e.g. vials andtransfer tubing) are sterile. The components and reagents can besterilised by methods known in the art, including: sterile filtration,terminal sterilisation using e.g. gamma-irradiation, autoclaving, dryheat or chemical treatment (e.g. with ethylene oxide). It is preferredto sterilise the non-radioactive components in advance, so that theminimum number of manipulations need to be carried out on theradioiodinated radiopharmaceutical product. As a precaution, however, itis preferred to include at least a final sterile filtration step.

The precursors of Formula (IA) or (IB), and other reactants, reagentsand solvents are each supplied in suitable vials or vessels whichcomprise a sealed container which permits maintenance of sterileintegrity and/or radioactive safety, plus optionally an inert headspacegas (eg. nitrogen or argon), whilst permitting addition and withdrawalof solutions by syringe or cannula. A preferred such container is aseptum-sealed vial, wherein the gas-tight closure is crimped on with anoverseal (typically of aluminium). The closure is suitable for single ormultiple puncturing with a hypodermic needle (e.g. a crimped-on septumseal closure) whilst maintaining sterile integrity. Such containers havethe additional advantage that the closure can withstand vacuum ifdesired (eg. to change the headspace gas or degas solutions), andwithstand pressure changes such as reductions in pressure withoutpermitting ingress of external atmospheric gases, such as oxygen orwater vapour. The reaction vessel is suitably chosen from suchcontainers, and preferred embodiments thereof. The reaction vessel ispreferably made of a biocompatible plastic (eg. PEEK).

When the radioiodinated fatty acid is used as a pharmaceuticalcomposition, the method of the second or third aspects is preferablycarried out using an automated synthesizer apparatus. By the term“automated synthesizer” is meant an automated module based on theprinciple of unit operations as described by Satyamurthy et at [Clin.Positr. Imag., 2(5), 233-253 (1999)]. The term ‘unit operations’ meansthat complex processes are reduced to a series of simple operations orreactions, which can be applied to a range of materials. Such automatedsynthesizers are preferred for the method of the present inventionespecially when a radiopharmaceutical product is desired. They arecommercially available from a range of suppliers [Satyamurthy et al,above], including: GE Healthcare; CTI Inc; Ion Beam Applications S.A.(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest(Germany) and Bioscan (USA).

Commercial automated synthesizers also provide suitable containers forthe liquid radioactive waste generated as a result of theradiopharmaceutical preparation. Automated synthesizers are nottypically provided with radiation shielding, since they are designed tobe employed in a suitably configured radioactive work cell. Theradioactive work cell provides suitable radiation shielding to protectthe operator from potential radiation dose, as well as ventilation toremove chemical and/or radioactive vapours. The automated synthesizerpreferably comprises a cassette.

By the term “cassette” is meant a piece of apparatus designed to fitremovably and interchangeably onto an automated synthesizer apparatus(as defined below), in such a way that mechanical movement of movingparts of the synthesizer controls the operation of the cassette fromoutside the cassette, i.e. externally. Suitable cassettes comprise alinear array of valves, each linked to a port where reagents or vialscan be attached, by either needle puncture of an inverted septum-sealedvial, or by gas-tight, marrying joints. Each valve has a male-femalejoint which interfaces with a corresponding moving arm of the automatedsynthesizer. External rotation of the arm thus controls the opening orclosing of the valve when the cassette is attached to the automatedsynthesizer. Additional moving parts of the automated synthesizer aredesigned to clip onto syringe plunger tips, and thus raise or depresssyringe barrels.

The cassette is versatile, typically having several positions wherereagents can be attached, and several suitable for attachment of syringevials of reagents or chromatography cartridges (eg. solid phaseextraction, SPE). The cassette always comprises a reaction vessel. Suchreaction vessels are preferably 1 to 10 cm³, most preferably 2 to 5 cm³in volume and are configured such that 3 or more ports of the cassetteare connected thereto, to permit transfer of reagents or solvents fromvarious ports on the cassette. Preferably the cassette has 15 to 40valves in a linear array, most preferably 20 to 30, with 25 beingespecially preferred. The valves of the cassette are preferably eachidentical, and most preferably are 3-way valves. The cassettes of thepresent invention are designed to be suitable for radiopharmaceuticalmanufacture and are therefore manufactured from materials which are ofpharmaceutical grade and ideally also are resistant to radiolysis.

Preferred automated synthesizers of the present invention are thosecomprising a disposable or single use cassette which comprises all thereagents, reaction vessels and apparatus necessary to carry out thepreparation of a given batch of radioiodinated radiopharmaceutical. Thecassette means that the automated synthesizer has the flexibility to becapable of making a variety of different radioiodine-labelledradiopharmaceuticals with minimal risk of cross-contamination, by simplychanging the cassette. The cassette approach also has the advantages of:simplified set-up hence reduced risk of operator error; improved GMP(Good Manufacturing Practice) compliance; multi-tracer capability; rapidchange between production runs; pre-run automated diagnostic checking ofthe cassette and reagents; automated barcode cross-check of chemicalreagents vs the synthesis to be carried out; reagent traceability;single-use and hence no risk of cross-contamination, tamper and abuseresistance.

In a fourth aspect, the present invention provides a radiopharmaceuticalcomposition comprising an effective amount of the radioiodinated fattyacid of Formula (I) as defined in the first aspect, together with abiocompatible carrier medium.

Preferred embodiments of the radioiodinated fatty acid of Formula (I) inthe fourth aspect are as defined in the first aspect.

The “biocompatible carrier medium” comprises one or morepharmaceutically acceptable adjuvants, excipients or diluents. It ispreferably a fluid, especially a liquid, in which the radioiodinatedfatty acid of Formula (I) is suspended or dissolved, such that thecomposition is physiologically tolerable, i.e. can be administered tothe mammalian body without toxicity or undue discomfort. Thebiocompatible carrier medium is suitably an injectable carrier liquidsuch as sterile, pyrogen-free water for injection; an aqueous solutionsuch as saline (which may advantageously be balanced so that the finalproduct for injection is either isotonic or not hypotonic); an aqueoussolution of one or more tonicity-adjusting substances (eg. salts ofplasma cations with biocompatible counterions), sugars (e.g. glucose orsucrose), sugar alcohols (eg. sorbitol or mannitol), glycols (eg.glycerol), or other non-ionic polyol materials (eg. polyethyleneglycols,propylene glycols and the like). The biocompatible carrier medium mayalso comprise biocompatible organic solvents such as ethanol. Suchorganic solvents are useful to solubilise more lipophilic compounds orformulations. Preferably the biocompatible carrier medium ispyrogen-free water for injection, isotonic saline or an aqueous ethanolsolution. The pH of the biocompatible carrier medium for intravenousinjection is suitably in the range 4.0 to 10.5.

In a fifth aspect, the present invention provides the use of theprecursor of Formula (IA) as defined in the second aspect, or theprecursor of Formula (IB) as defined in the third aspect in themanufacture of the radioiodinated fatty acid of Formula (I) as definedin the first aspect, or for the manufacture of the radiopharmaceuticalcomposition of the fourth aspect.

Preferred embodiments of the radioiodinated fatty acid of Formula (I),precursor of Formula (IA) or of Formula (IB) in the use of the fifthaspect, are as defined in the first, second and third aspectsrespectively.

In a sixth aspect, the present invention provides the use of anautomated synthesizer apparatus to carry out the method of preparationof the second or third aspects.

Preferred embodiments of the precursors, methods and automatedsynthesizer in the use of the sixth aspect are as described in thesecond and third aspects.

In a seventh aspect, the present invention provides a method ofgenerating an image of a human or animal body comprising administeringthe radioiodinated fatty acid of Formula (I) of the first aspect, or theradiopharmaceutical composition of the fourth aspect, and generating animage of at least a part of said body to which said compound orcomposition has distributed using PET or SPECT.

Preferred aspects of the radioiodinated fatty acid andradiopharmaceutical composition in the seventh aspect are as describedin the first and fourth aspects respectively.

The radioiodinated fatty acids of the invention are useful for imagingmyocardial metabolism, in particular for patients with coronary arterydisease. Such imaging includes the imaging of: acute myocardialinfarction; unstable angina; myocardial viability assessment andprediction of recovery of function in chronic coronary artery disease;risk stratification and prognosis. The agent may also be useful, inconjunction with myocardial perfusion assessment for patients withcardiomyopathy. Further details are provided by Taki et al [Eur. J.Nucl. Med. Mol. Imaging, 34, S34-S48 (2007)].

In a further aspect, the present invention provides a method ofmonitoring the effect of treatment of a human or animal body with adrug, said method comprising administering to said body theradioiodinated fatty acid of Formula (I) as defined in the first aspect,or the radiopharmaceutical composition of the fourth aspect, anddetecting the uptake of said fatty acid or composition in at least apart of said body to which said fatty acid or composition hasdistributed using PET or SPECT, said administration and detectionoptionally but preferably being effected before, during and aftertreatment with said drug. The administration and detection of this finalaspect are preferably effected before and after treatment with saiddrug, so that the effect of the drug treatment on the human or animalpatient can be determined. Where the drug treatment involves a course oftherapy, the imaging can also be carried out during the treatment. Thediseases or conditions being treated in the further aspect are asdescribed in the seventh aspect (above)

The invention is illustrated by the following Examples. Example 1provides the synthesis of ¹²³I-iodoacetylene. Example 2 provides theclick cycloaddition of ¹²³I-iodoacetylene to an azide derivative, toform a radioiodinated triazole ring. Example 3 provides the clickcycloaddition of ¹²³I-iodoacetylene to an isonitrile oxide derivative,to form a radioiodinated isoxazole ring. Example 4 provides a clickcycloaddition of a tributyltin-alkyne to an azide derivative, to form atriazole radioiodination precursor having a triazole-tributyltin bond.Example 5 provides the conditions for converting the precursor ofExample 4, to the radioiodinated product. Example 6 provides a synthesisof an isoxazole radioiodination precursor having anisoxazole-tributyltin bond via click cycloaddition from an isonitrileoxide derivative. Example 7 provides the radioiodination of theprecursor of Example 6. Example 8 provides the synthesis of aniodotriazole-substituted fatty acid. Example 9 provides the synthesis ofan iodoisoxazole-substituted fatty acid.

Abbreviations Used in the Examples

HPLC: high performance liquid chromatography,PAA: peracetic acid,RCP: radiochemical purity,THF: tetrahydrofuran.

Example 1 Preparation and Distillation of [¹²³I]-Iodoacetylene UsingPeracetic Acid Oxidant

To a Wheaton vial on ice was added, ammonium acetate buffer (100 μl, 0.2M, pH 4), sodium [¹²⁷I] iodide (10 μl, 10 mM solution in 0.01M sodiumhydroxide, 1×10⁻⁷ moles), sodium [¹²³I] iodide (20 μl, 53 MBq),peracetic acid, (10 μl, 10 mM solution, 1×10⁻⁷ moles) and a solution ofethynyltributylstannane in THF (Sigma-Aldrich; 38 μl, 1 mg/mL, 1.2×10⁻⁷moles). Finally, 460 μl THF was added, the Wheaton vial sealed and thereaction mixture allowed to warm to room temperature prior to reversephase HPLC analysis which showed [¹²³I]-iodoacetylene with aradiochemical purity (RCP) of 75% (t_(R) 12.3 minutes, System A).

The reaction mixture was heated at 80-100° C. for 30 minutes duringwhich time, the [¹²³I]-iodoacetylene and THF were distilled through ashort tube into a collection vial on ice. After this time, a low flow ofnitrogen was passed through the septa of the heated vial to remove anyresidual liquids from the tube. [¹²³I]-iodoacetylene was collected in38.6% yield (non decay corrected) with an RCP of 94%. (t_(R) 12.3minutes, System A).

HPLC System A (A=water; B=acetonitrile).

Column C18 (2) phenonenex Luna, 150×4.6 mm, 5 micron

Gradient Time (min) 0 1 20 25 25.5 30 % B 5 5 95 95 5 5

Example 2 Preparation of 1-Benzene-4-[¹²³I]-iodo-1H-1,2,3 triazole(Prophetic Example)

To a Wheaton vial charged with copper powder (200 mg, −40 mesh), sodiumphosphate buffer (200 mL, pH 6, 50 mM) and placed on ice is added,[¹²³I]-iodoacetylene and benzyl azide (1 mg, 7.5×10⁻⁶ moles). Followingreagent addition, the ice bath is removed and the reaction incubated atroom temperature with heating applied as required.1-Benzene-4-[¹²³I]-iodo-1H-1,2,3-triazole is purified by reverse phaseHPLC.

Example 3 Preparation of 5-[¹²³I]-iodo-3-phenyl isoxazole (PropheticExample)

To a Wheaton vial charged with copper powder (50 mg, −40 mesh), copper(II) sulphate (3.8 μg, 1.53×10-8 moles, 0.5 mg/mL solution in water),sodium phosphate buffer (100 μL, 50 mM, pH6) and placed on ice, is added[¹²³I]-iodoacetylene and benzonitrile-N-oxide (1 mg, 8.4×10⁻⁶ moles.Following reagent addition, the ice bath is removed and the reactionincubated at room temperature with heating applied as required.5-[¹²³I]-iodo-3-phenyl isoxazole is purified by reverse phase HPLC.

Example 4 Preparation of 1-Phenyl-4-(tributylstannyl)-1H [1,2,3]triazole (Prophetic Example)

Phenylazide can be obtained from Sigma-Aldrich or can be synthesized bythe method described in J. Biochem., 179, 397-405 (1979). A solution oftributylethynyl stannane (Sigma Aldrich; 400 mg, 1.27 mmol) in THF (4mL) is treated with phenylazide (169 mg, 1.27 mmol), copper (I) iodide(90 mg, 0.47 mmol), and triethylamine (256 mg, 2.54 mmol) at roomtemperature over 48 h. The reaction is then filtered through celite toremove copper (I) iodide and chromatographed on silica in a gradient of5-20% ethyl acetate in petrol. The second fraction is collected andconcentrated in vacuo to give the 1-phenyl-4-(tributylstannyl)-1H[1,2,3] triazole as a colourless oil.

Example 5 Preparation of [¹²³I]-Phenyl-4-iodo-1H [1,2,3] triazole UsingPeracetic Acid as the Oxidant (Prophetic Example)

To sodium [¹²³I] iodide, received in 5-20 μL 0.05M sodium hydroxide isadded ammonium acetate buffer (100 μL pH 4.0, 0.2M), sodium [¹²⁷I]iodide (10 μL 1 mM solution in 0.01M sodium hydroxide, 1×10⁻⁸ moles),peracetic acid (PAA) solution (10 μL 1 mM solution, 1×10⁻⁸ moles) andfinally, 1 phenyl-4-tributylstannyl-1H [1,2,3] triazole (Example 4; 43μg, 1×10⁻⁷ moles) dissolved in acetonitrile. The reaction mixture isincubated at room temperature for 15 minutes prior to purification byHPLC.

Example 6 Preparation of 3-Phenyl-5-(tributylstannyl) isoxazole(Prophetic Example)

(E)-benzaldehyde oxime (Sigma Aldrich; 3.3 g, 20 mmol) in tert butanoland water (1:1) 80 mL, is treated with chloramine T trihydrate (SigmaAldrich; 5.9 g, 21 mmol) in small, portions over 5 min. The reaction isthen treated with copper sulfate pentahydrate (0.15 g, 0.6 mmol) andcopper turnings ˜50 mg and tributylethynylstannane (6.3 g, 20 mmol). Thereaction is then adjusted to pH 6 with sodium hydroxide solution andstirred for 6 h. The reaction mixture is treated with dilute ammoniumhydroxide solution to remove all copper salts. The product is collectedby filtration, redissolved in ethyl acetate and filtered through a shortplug of silica gel. The filtrate is concentrated in vacuo to give3-phenyl-5-(tributylstannyl) isoxazole.

Example 7 Preparation of 5-[¹²³I]-Iodo-3-phenyl isoxazole (PropheticExample)

To sodium [¹²³I] iodide, received in 5-20 μL 0.05M sodium hydroxide isadded ammonium acetate buffer (100 μL pH 4.0, 0.2M), sodium [¹²⁷I]iodide (10 μL, 1 mM solution in 0.01M sodium hydroxide, 1×10⁻⁸ moles),peracetic acid (PAA) solution (10 μL 1 mM solution, 1×10⁻⁸ moles) andfinally, 3-phenyl-5-tributylstannyl-isoxazole (Example 6; 43 μg, 1×10⁻⁷moles) dissolved in acetonitrile. The reaction mixture is incubated atroom temperature for 15 minutes prior to purification by HPLC.

Example 8 Preparation of 10-(4-Iodo-[1,2,3]triazol-1-yl)-decanoic acid(Prophetic Example) Step (a): Preparation of10-(4-azido-[1,2,3]triazol-1-yl)-decanoic acid

10-Bromodecanoic acid (2.51 g, 10 mmol) in acetone (50 mL) is treatedwith sodium azide (0.75 g, 11 mmol) at reflux for 2 h. The reaction isthen concentrated in vacuo to a gum that is partitioned between ethylacetate (50 mL) and water (50 mL). The organic phase is separated, driedover sodium sulfate and concentrated in vacuo to give 10-azidodecanoicacid (2.02 g, 95%).

Step (b): Preparation of10-(4-tributylstannyl-[1,2,3]triazol-1-yl)-decanoic acid

10-Azidodecanoic acid (2.0 g, 9.5 mmol) in THF (50 mL) is treated withtributylstannyl acetylene (2.99 g, 9.5 mmol) copper(I)iodide (90 mg 0.47mmol) and triethylamine (256 mg, 2.54 mmol) at room temperature withconstant stirring for 24 h. The reaction is then filtered through celiteand concentrated in vacuo to a gum, and then chromatographed on silicain a gradient of 10-40% ethyl acetate in petrol. The main fraction iscollected and concentrated in vacuo to give10-(4-tributylstannyl-[1,2,3]triazol-1-yl)-decanoic acid (3.22 g, 0.8mmol).

Step (c): Preparation of 10-(4-iodo-[1,2,3]triazol-1-yl)-decanoic acid

To sodium [¹²³J] iodide, received in 5-20 μL 0.05M sodium hydroxide isadded ammonium acetate buffer (100 μL pH 4.0, 0.2M), sodium [¹²⁷J]iodide (10 μL, 1 mM solution in 0.01M sodium hydroxide, 1×10⁻⁸ moles),peracetic acid (PAA) solution (10 μL 1 mM solution, 1×10⁻⁸ moles) andfinally 10-(4-tributylstannyl-[1,2,3]triazol-1-yl)-decanoic acidsolution in ethanol or acetonitrile (53 μg, 1×10⁻⁷ moles). The reactionmixture is allowed to stand at room temperature for 15 minutes prior toHPLC purification of the iodinated product10-(4-iodo-[1,2,3]triazol-1-yl)-decanoic acid.

Example 9 Preparation of 10-(5-Iodo isoxazol-3-yl)-decanoic acid(Prophetic Example) Step (a): Preparation of 11[(E)-hydroximino-decanoicacid

A solution of dimethyl sulfoxide (1.17 g, 15 mmol) in dichloromethane(50 mL) is cooled to −30° C. and treated with oxalyl chloride (1.92 g,15 mmol). The reaction mixture is then treated with 10-hydroxydecanoicacid (2.06 g, 10 mmol) in dichloromethane (50 mL) and allowed to warm toroom temperature over a period of 2 h. The reaction is then washed withwater (2×50 mL). The organic layer is dried over sodium sulfate and thentreated with hydroxylamine hydrochloride (1.03 g 15 mmol) and sodiumhydroxide (0.6 g in 10 mL water) and stirred vigorously for 1 h. Theorganic phase is then separated dried over sodium sulfate andconcentrated in vacuo to give 11[(E)-hydroxyimino-decanoic acid (1.8 g,9.0 mmol).

Step (b): Preparation of 10-(5-tributylstanyl-isoxazol-3-yl)-decanoicacid

11[(E)-Hydroxyimino-decanoic acid (1.8 g, 9.0 mmol) is dissolved inacetonitrile (30 ml) and the solution cooled to 0° C. and then treatedwith a solution of chloramine-T trihydrate (2.52 g, 9.0 mmol) in water(10 mL). The reaction is allowed to warm to room temperature over aperiod of 25 minutes. To the resulting nitrile oxide solution is addedtributyl(ethynyl)stannane (2.83 g, 9.0 mmol), copper iodide (100 mg, 0.5mmol) and triethylamine (50 mg, 0.5 mmol) and the reaction stirred atroom temperature for 24 h. The reaction is then filtered through celiteto remove the copper salts and concentrated in vacuo to a gum. The gumis then chromatographed on silica in a gradient of 10- 30% ethyl acetatein petrol to give 10-(5-tributylstanyl-isoxazol-3-yl)-decanoic acid.

Step (c) Example 4 Preparation of 10-(5-iodo isoxazol-3-yl)-decanoicacid

To sodium [¹²³I] iodide, received in 5-20 μL 0.05M sodium hydroxide isadded ammonium acetate buffer (100 μL pH 4.0, 0.2M), sodium [¹²⁷I]iodide (10 μL, 1 mM solution in 0.01M sodium hydroxide, 1×10⁻⁸ moles),peracetic acid (PAA) solution (10 μL 1 mM solution, 1×10⁻⁸ moles) andfinally 10-(5-tributylstannyl-isoxazol-3-yl)-decanoic acid solution inethanol or acetonitrile (53 μg, 1×10⁻⁷ moles). The reaction mixture isallowed to stand at room temperature for 15 minutes and the iodinatedproduct 10-(4-iodo-[1,2,3]triazol-1-yl)-decanoic acid purified by HPLC.

1-18. (canceled)
 19. A radioiodinated fatty acid of Formula (I):

where: R¹ and R² are independently H or C₁₋₂ alkyl; Y is a Y² group:

p and q are each independently integers of value 0 to 10 which are chosen such that [p+q] is in the range 10 to 16; L¹ is a linker group of formula -(A)_(n)- where n is an integer of value 0 to 3, and each A group is independently chosen from —CH₂—, —O—, —S— and —C₆H₄— with the proviso that L¹ does not comprise —O—O—, —S—S— or —O—S— linkages; and I* is a radioisotope of iodine.
 20. The radioiodinated fatty acid of claim 19, wherein I* is chosen from ¹²³I, ¹²⁴I or ¹³¹I.
 21. The radioiodinated fatty acid of claim 19, where R¹ is CH₃ and R² is H.
 22. The radioiodinated fatty acid of claim 19, where L¹ is chosen from —CH₂—, —O— and —S—.
 23. The radioiodinated fatty acid of claim 19, where n=0, and [p+q] is in the range 11 to
 13. 24. A radiopharmaceutical composition comprising an effective amount of the radioiodinated fatty acid of Formula (I) as defined in claim 19, together with a biocompatible carrier medium. 